Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Production of a cytotoxic cationic antibacterial peptide in Escherichia coli using SUMO fusion partner


Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide ABP-CM4. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 112 mg protein of interest was obtained per liter of fermentation culture. After the SUMO–CM4 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 24 mg recombinant CM4 was obtained from 1 l fermentation culture with no less than 96% purity and the recombinant CM4 had similar antimicrobial properties to the synthetic CM4. Thus, the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant cytotoxic peptides.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Butt TR, Edavettal SC, Hall JP, Mattern MR (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43:1–9

  2. Chen YQ, Zhang SQ, Li BC, Qiu W, Jiao B, Zhang J, Diao ZY (2008) Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners. Protein Expr Purif 57:303–311

  3. Hammarstrom M, Hellgren N, van Den Berg S, Berglund H, Hard T (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci 11:313–321

  4. Hancock RE (2001) Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 1:156–164

  5. Kamysz W, Okroj M, Lukasiak J (2003) Novel properties of antimicrobial peptides. Acta Biochim Pol 50:461–469

  6. Koczulla AR, Bals R (2003) Antimicrobial peptides: current status and therapeutic potential. Drugs 63:389–406

  7. Lehrer RI, Rosenman M, Harwig SL, Jackson R, Eisenhauer P (1991) Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods 137:167–173

  8. Li P, Zhinan X, Xiangming F, Fang W, Peilin C (2004) High-level expression of soluble human Beta-Defensin-2 in E. coli. Process Biochem 39:2199–2205

  9. Li Y, Li X, Wang G (2006) Cloning expression isotope labeling and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies. Protein Expr Purif 47:498–505

  10. Li BC, Zhang SQ, Dan WB, Chen YQ, Cao P (2007) Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori. Biotechnol Lett 29:1031–1036

  11. Ma XY, Zheng WY, Wei DZ, Ma YS, Wang TW, Wang JZ, Liu QH, Yang SL (2006) High-level expression, purification and pro-apoptosis activity of HIV-TAT-survivin (T34A) mutant to cancer cells in vitro. J Biotechnol 123:367–378

  12. Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15:182–189

  13. Pyo SH, Lee JH, Park HB, Cho JS, Kim HR, Han BH, Park YS (2004) Expression and purification of a recombinant buforin derivative from Escherichia coli. Proc Biochem 39:1731–1736

  14. Rao XC, Li S, Hu JC, Jin XL, Hu XM, Huang JJ, Chen ZJ, Zhu JM, Hu FQ (2004) A novel carrier molecule for high-level expression of peptide antibiotics in Escherichia coli. Protein Expr Purif 36:11–18

  15. Robert WK, Selitrennikoff CP (1986) Isolation and partial characterization of two antifungal proteins from barley. Biochim Biophys Acta 880:161–170

  16. Skosyrev VS, Rudenko NV, Yakhnin AV, Zagranichny VE, Popova LI, Zakharov MV, Gorokhovatsky AY, Vinokurov LM (2003) EGFP as a fusion partner for the expression and organic extraction of small polypeptides. Protein Expr Purif 27:55–62

  17. Sun Z, Xia Z, Bi F, Liu JN (2008) Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli. Appl Microbiol Biotechnol 78:495–502

  18. Xu Z, Zhong Z, Huang L, Peng L, Wang F, Cen P (2006) High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression. Appl Microbiol Biotechnol 72:471–479

  19. Yizeng T, Zhang S, Qu X (1989) Separation, purification of antibacterial CM4 and the research of the structure and character. Sci China B 32:473–480

  20. Zhang SQ, Jia HW, Dai ZY (1997) Ultrastructure observation of K562 leukemia cells treated with antibacterial peptide CM4 component. Prog Biochem Biophys 24:159–163

  21. Zhang L, Falla T, Wu M, Fidai S, Burian J, Kay W, Hancock REW (1998) Determinants of recombinant production of antimicrobial cationic peptides and creation of peptide variants in bacteria. Biochem Biophys Res Commun 247:674–680

  22. Zhou LF, Zhao ZH, Li BC, Cai YF, Zhang SQ (2009) TrxA mediating fusion expression of antimicrobial peptide CM4 from multiple joined genes in Escherichia coli. Protein Expr Purif 64:225–230

  23. Zuo X, Li S, Hall J, Mattern MR, Tran H, Shoo J, Tan R, Weiss SR, Butt TR (2005) Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli. J Struct Funct Genomics 6:103–111

Download references


This work was supported by the Grants of Nanjing Normal University and Jiangsu Province Graduate Innovation Project (No. CX07S-020z) administered by Prof. Zhang.

Author information

Correspondence to Shuang Quan Zhang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, J.F., Zhang, J., Song, R. et al. Production of a cytotoxic cationic antibacterial peptide in Escherichia coli using SUMO fusion partner. Appl Microbiol Biotechnol 84, 383–388 (2009).

Download citation


  • Antibacterial peptide
  • ABP-CM4
  • SUMO
  • Escherichia coli